Binary or polynary targeting and uses thereof

ABSTRACT

Effector functions are provided to a desired target with improved specificity by use of two or more complementary targeting components. The targeting components assemble a functional moiety at the target. At the target, the functional moiety itself provides an effector function or binds to additional components which provide an effector function. The effector function may be an enzymatic activity, a label or a signal. The binary or polynary targeting system may be used for analyte determination as well.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/377,067, filed May 1, 2002. The contents of thisapplication are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to targeting of effectorfunctions to a chosen site with improved specificity based on use of atleast binary targeting components, and uses thereof. More specifically,the invention provides methods and kits for cellular targeting whichrely on the assembly at the targeted site of a functional moiety fromtwo or more independently targeted components. A variety of effectorfunctions can thereby be provided at the chosen site with higherspecificity than is possible for the same effector functions deliveredby a single targeting component.

BACKGROUND ART

[0003] It has long been recognized that it is, in many cases, desirableto provide a targeted site with a property or function to the exclusionof the environment which surrounds this site. Early examples of suchspecific targeting include the design of immunotoxins where, inprinciple, a toxic moiety could be delivered selectively andspecifically to a cellular target where the toxicity was desired withoutnegative side effects on the surrounding tissue. It may also bedesirable, as further described below, to provide an enzymatic activityat a particular location to the exclusion of the surroundings.Typically, attempts have been made to accomplish this by using a singleligand to carry the active principle to the desired location, relying onthe affinity of the ligand for the targeted site to provide selectivity.

[0004] However, in many instances, this approach does not confersufficient selectivity. For example, it has been established that thepharmacokinetics of antibody binding to tumor cells is slow (24-48 hoursfor maximal specificity). During this time, a toxic effector coupled tothe antibody would be expected to have deleterious effects on theremainder of the system. Coupling the antibody against a tumor cellsurface antigen to avidin, has been suggested as a way to avoid systemicexposure to toxic effectors. Administering a biotin conjugated toxin orradioligand after the antibody is maximally localized results inclearing of the small molecule from the body in less than one hour,thereby greatly reducing systemic exposure (Sung and van Osdol, J. Nucl.Med. (1995) 36(5):867-76).

[0005] Similarly, antibody-enzyme conjugates have been used to activatesmall molecule prodrugs administered after the antibody conjugate hasmaximally localized (Melton and Sherwood, J. Natl. Cancer Inst. (1996)88(3-4):153-65). This strategy is commercialized by Seattle Genetics,Inc. Enzymatic activation of a prodrug extracellularly is particularlydesirable for treating cancer because it creates a bystander effect forkilling tumor cells in the vicinity. Thus, the targeting antigen can beas simple as histones, present in large amounts in necrotic cell debriswithin most solid tumors.

[0006] Still another variation of the basic antibody-effector conjugateidea is exemplified by the TAP (tumor activated prodrug) technologycommercialized by Immunogen, in which an antibody conjugated to a toxin,e.g., maytansine, is internalized preferentially by cells expressing therelevant antigen for that antibody, with toxin released intracellularly(Liu and Chari, Exp. Opi. Invest. Drugs (1997) 6:169-172). A drawback ofthe TAP approach is that it lacks a bystander effect, thus selecting formutants that have lost the targeting antigen.

[0007] All of these existing techniques suffer from the intrinsic limitson specificity of the targeting antibody. In this aspect, target cellsare typically a small fraction of all cells in the body, often in theneighborhood of 1/100,000 or less. Accordingly, even if the antibody hasvery high specificity, with affinity for target antigen being 100,000times higher than for any other antigen in the body, the fraction of theconjugated effector that is distributed to non-target cells is still50%. In practice, antibody specificities are not usually this high, andthe background binding is correspondingly higher. Thus, in this context,the use of a single targeting ligand is generally less thansatisfactory.

[0008] The goal of the present invention is to improve the specificityof targeting in general by requiring multiple independent binding eventsbefore an effector function is created. This approach is not limited todelivering effector functions to the surface of cells in vivo, but canalso be used to deliver effector functions to intracellular targets, andto cellular or non-cellular targets in vitro.

[0009] The invention also provides specific methods and kits for binaryor polynary targeting to create an enzymatic activity, of particularutility for activating prodrugs at cell surfaces. The invention furtherprovides methods and kits for assaying an analyte using binary orpolynary targeting.

[0010] A natural example for binary reconstitution of an effector is thegramicidin toxin, which creates a pore through the cell membrane by endto end dimerization of an ion channel that spans half the width of themembrane. In nature, this particular toxin can flip in the plane of themembrane so it is not necessary to target one monomer to the inside andone to the outside of the cell in order to create the effector function.Although these precedents suggest that the present invention may beembodied in a naturally existing construct, it is normally necessary toalter and/or combine several moieties to make a targeting component withall the requisite properties. The present invention excludes suchnaturally occurring phenomena.

DISCLOSURE OF THE INVENTION

[0011] This invention relates generally to targeting of effectorfunctions to a desired site by means of multiple targeting components.The present method can be used therapeutically, as well as forprognostic and diagnostic monitoring, and for basic research in cellbiology. The invention requires at least two targeting components andbinary targeting is the preferred embodiment; however, three or moretargeting components could also be used. Binary targeting is used inmany instances for illustration, without thereby limiting the inventionto the binary embodiment.

[0012] In one aspect, the present invention provides a method to createan effector function selectively at a desired location. The method canbe used, e.g., for visualizing the target, for delivering a drug to thetarget, for detecting presence of competing analytes, for creating anenzymic activity, and for many other applications. An effector functionis created by assembly of individually inactive moieties. Thefundamental feature of all embodiments is the provision of two or moretargeting components, each of which comprises a targeting portion and areconstitution portion, wherein the targeting portions bind specificallyto distinct sites located in close proximity at the target and whereinthe reconstitution portions, when brought into close proximity, assembleinto a functional moiety.

[0013] The functional moiety may itself provide an effector function,such as an enzymic activity or toxicity. -Alternatively, the functionalmoiety may provide the effector function indirectly by binding to one ormore additional materials, such as a toxin or an enzyme.

[0014] Thus, in one aspect, the invention is directed to a method toprovide a functional moiety specifically to a target which methodcomprises contacting an environment containing the target with at leasttwo targeting components wherein each of said targeting componentscomprises a targeting portion and a reconstitution portion. Eachtargeting portion binds specifically to one of two or more sites locatedin close proximity on the target. The reconstitution portions, whenbrought into close proximity, assemble into a functional moiety. Asdescribed above, the functional moiety may itself provide an effectivefunction or may bind to one or more additional components which providesuch functions.

[0015] This method of the invention is adaptable to a variety ofapplications, including use in vivo to deliver drugs, labels, toxins, ordesired components in general to specific targets, use in vitro for thesame purpose, and specifically, in a method to assay for an analytewhich has the ability to interfere with the formation of the functionalmoiety.

[0016] In another aspect, the present invention discloses a kit toprovide a functional moiety, and ultimately an effector function to atarget, which kit comprises, in a container, two or more targetingcomponents, each of which comprises a targeting portion and areconstitution portion. If the reconstitution portions do not generatedirectly an effector function, additional components which interact withthe functional moiety formed by the reconstitution portions may also beincluded. Preferably, the kit further comprises instructions for usingthe targeting components and/or the effector to provide an effectorfunction to a cellular structure or other chosen site.

[0017] In yet another aspect, the present invention provides a methodfor competitively assaying an analyte in a sample by assessing theability of the analyte to interfere with the formation of the functionalmoiety, for example by to preventing one of the targeting componentsfrom reaching the site at which its complementary targeting component islocated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings are supplied to assist in understanding theinvention, and are not intended to be complete or to limit the scope.

[0019]FIG. 1 illustrates the basic concept of the invention. Sites inclose proximity (a, b) bind targeting components (c), which comprise atargeting portion (d), an optional linker (e), and a reconstitutionportion (f), wherein the reconstitution portions assemble into afunctional moiety (g) when brought into close proximity by virtue of thetargeting portions binding to the sites in close proximity.

[0020] FIGS. 2(a)-2(f) illustrate several formats by whichreconstitution portions (1 and 2) can assemble into a functional moiety.

[0021]FIG. 2(a): the assembled reconstitution portions provide aneffector function that has enzymatic activity on substrate (3).

[0022]FIG. 2(b): reconstitution portion (1) modulates the enzymaticactivity of reconstitution portion (2) on substrate (3), by supplying anallosteric activator.

[0023]FIG. 2(c): the assembled functional moiety binds a fluorescentligand (3 ¹) wherein that binding stimulates emission.

[0024]FIG. 2(d): the assembled functional moiety binds an epitope (3 ²)present on an enzyme (6) naturally, or by epitope tagging, or coupled tothe enzyme via an optional linking pair (4 and 5).

[0025]FIG. 2(e): the assembled moiety functions as a receptor for avirus (3 ³).

[0026]FIG. 2(f): the assembled moiety constitutes a discontinuousepitope recognized by an antibody (3 ⁴) conjugated to an enzyme (4).

MODES OF CARRYING OUT THE INVENTION

[0027] The basic principle of the invention is that independentlocalization of two or more targeting portions at a single structure canbe used to bring attached reconstitution components into molecular scaleproximity as needed to form a functional moiety that can itself carryout an effector function or which can couple to additional component(s)to provide an effector function. The combination of a targeting portionand a reconstitution portion constitutes a targeting component.

[0028] To be used in the present method, a targeting component must havethe following three properties. First, each targeting component mustpossess a targeting portion to direct the component to the chosen site,either from outside or inside the cell or in vitro. Second, eachtargeting component must possess, as a reconstitution portion, one pieceof a functional moiety, wherein that piece alone does not create thedesired function by itself. Third, the targeting components must becapable of assembling a functional moiety at the desired target.

[0029] The targeting portions will normally bind to sites in closeproximity, with spacing of 5 to 50 nm being preferred. Such sites can bepresent on a cell surface or on an artificial surface such as a 96-wellplastic microplate. The targeting components can also be providedintracellularly, for example using nucleic acid vectors that encode thetargeting components.

[0030] Targeting components whose targeting portions bind to sites inclose proximity at a target are referred to as “complementary”. Thecomplementary targeting components may comprise two such components,three such components or four or more such components depending on thenature of the reconstitution portions and location and nature of theproximal sites.

[0031] As will be described in more detail below, suitable moieties foruse as targeting portions include antibodies, peptides,oligonucleotides, carbohydrates, and other biospecific moieties.Likewise, as detailed below, a wide range of chemical entities canparticipate as reconstitution portions, including proteins,oligonucleotides, and vitamins. “Reconstitution portion” means a moietythat by itself does not provide a desired functionality but which cancontribute to a moiety providing that functionality when brought intoclose proximity to one or more complementary reconstitution portions.The assembled “functional moiety” can provide an “effector function” byitself. For example, the reconstitution portions can provide anenzymatic activity when assembled. Alternatively, the reconstitutionportions can provide a functional moiety when assembled to which aneffector can bind. In contrast to the targeting portions, a requirementfor each of the reconstitution portions is that it cannot provide thedesired function by itself. Rather, two or more reconstitution portionsmust be brought into close proximity to assemble into a functionalmoiety. For example, in a preferred embodiment, the variable regions oflight and heavy chains of an antibody are used as reconstitutionportions, each representing a “demitope” (half of an antigen bindingdomain, which is known as a “paratope” in the immunological literature).The demitopes are selected such that neither alone can bind a chosenepitope at appreciable affinity, but when brought together, they do forma functional paratope able to bind the chosen epitope.

[0032] The targeting portion and the reconstitution portion, whichtogether form a targeting component, can be linked in any suitablemanner. A covalent linkage is preferred although sufficiently highaffinity non-covalent linkage can also be used. The targeting portionand the reconstitution portion can be prepared separately and thenchemically linked, directly or via a short linker. Alternatively, atleast one of the targeting components is a fusion protein comprisingboth a targeting portion and a reconstitution portion.

[0033] The targeting components must bind to at least two sites locatedin distinct positions yet in sufficiently close proximity to enableassembly of a functional moiety. Such distinctly located sites can bethe same type, as found in a homodimeric protein for example, or theycan be different types, including non-overlapping epitopes on a largeprotein. The distinct sites can also be on separate molecules thatattain close proximity via diffusion, either laterally in the plane of amembrane or in three dimensions. Thus, for example, the necessaryproximity may be generated by patching of cell surface receptors. Asanother example, the necessary proximity may be generated by twoindependent molecules sharing a similar subcellular targeting property,e.g. by virtue of binding to the same promoter region on DNA or bygetting packaged into the same subcellular compartment such as aneurotransmitter synaptic vesicle.

[0034] Other terms which may benefit from formal definition are asfollows, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill inbiochemistry, augmented or extended by the following definitions, whichare intended to clarify the concepts without thereby limiting the scopeof the invention.

[0035] “Cellular structure” refers to an intact cell or to a subcellularstructure, e.g., nucleus, chromosome, mitochondrion, chloroplast,ribosome, endoplasmic reticulum, Golgi apparatus, lysosome, proteosome,secretory vesicle, vacuole, microsome, or virus. Cells include thosefrom animals, plants, fungi, bacteria, spores, including both naturaland recombinant cultured cells. A cellular structure can exist by itselfas a separated cell or subcellular structure, or it can exist as part ofa higher structure. For example, a subcellular structure can be part ofan intact cell, and an intact cell can be part of a multicellular tissueor organ or it can be present together with other cells of the same ordifferent types in a cell culture.

[0036] “Immunoglobulin” refers to proteins with sequence homology tocanonical immunoglobulin-like domains, i.e., a complex of heavy chainsand light chains each composed of a conserved scaffold and one or morevariable sequences. “Antibody” is a major type of immunoglobulin, andincludes the major forms familiar in the art such as IgG, IgM, IgE amongothers. However, an immunoglobulin can be a non-antibody molecule, suchas MHC molecules and some cell adhesion molecules and cytokinereceptors. An antibody moreover can exist in any suitable form, and asused here the term also encompasses any suitable fragments orderivatives. Exemplary antibodies under this wider definition include apolyclonal antibody, a monoclonal antibody, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, a Fv fragment, a diabody (two copies ofthe same Fv fragment fused), a single-chain antibody and amulti-specific antibody formed from more than one antibody fragment.Certain other standard immunological terminology will also be used in aslightly more general sense. Specifically, a “paratope” refers to theportion of an antibody that binds an antigenic determinant (an“epitope”); a “hapten” is a small molecule epitope, that is normally notimmunogenic in mammals unless conjugated to a carrier protein. In thepresent context, this definition of paratope and epitope extends toanalogous recognition elements on non-antibody moieties, includingreceptors and their ligands.

[0037] We have coined the term “demitope” to refer to complementaryportions of a paratope in this more general sense; demitope, then, isequivalent in meaning to reconstitution portion, but will generally beused here to highlight an immunological origin for the moiety.

[0038] Other antibody-like proteins can also be used to construct eitherthe targeting or reconstitution portions. As previously described by thepresent inventor and colleagues, it is feasible to mimic the propertiesof antibodies by embedding hypervariable regions, analogous toimmunoglobulin complementarity determining regions, in a scaffold basedon a protein other than the basic immunoglobulin scaffold. Specificallyglutathione transferase was shown to provide a suitable scaffold for afamily of proteins termed “glubodies” (Napolitano et al., Chem. Biol.(1996) 3(5):359-67). The use of “antibody” or “immunoglobulin” in thepresent disclosure, then, is meant to include not only conventional geneencoded IgG's and the like, but also glubodies, fragments of glubodies,and related constructs based on other protein scaffolds, as will bereadily apparent to one of ordinary skill in the field of proteinstructure and function.

[0039] “Gene therapy” refers to treatment of a disease by delivery tocells of a functional gene, directly or via a vector that includes afunctional gene.

[0040] “Prodrug” refers to a pharmaceutically inactive compound thatbecomes active within the body, either by means of natural chemicalchanges (e.g. induced by pH changes), or by enzymes normally present inthe body, or by enzymes introduced or engineered into the body.

[0041] “Exosite modulator” refers to a molecule whose binding to anenzyme, typically but not necessarily at a site spatially distinct fromthe enzyme's catalytic site, increases or decreases the activity of theenzyme. Allosteric regulators are a special case of this generalizedconcept, referring to naturally occurring exosite modulators. Similarly,the term exosite modulator for present purposes is intended to includecofactors, which are natural components of an enzyme that are notgenetically encoded in DNA but which form a normal part of the enzymaticreaction mechanism, such as metal ions, heme groups, redox or methylgroup donors, and the like.

[0042] “Epitope tagging” refers to a molecular biology technique inwhich the gene encoding a protein of interest is mutagenized to includean epitope for easy recognition, e.g. by an antibody. The protein ofinterest can then be analyzed, isolated or purified via biospecificrecognition of the epitope. Exemplary tags known in the art includepeptides derived from the myc gene, green fluorescent protein (GFP),haemagglutinin epitopes, and the FLAG peptide. Chemical attachment of ahapten serves the same purpose and for present purposes is also includedin the broader sense of epitope tagging.

[0043] Construction of Targeting Components

[0044] It is possible, although unlikely, that a naturally occurringsubstance may possess all these properties and thus could be used as atargeting component. For example, the toxin ricin consists of twoproteins, one that poisons ribosomes and one that binds a cell surfacelectin, promoting cell entry. The two proteins are linked by a disulfidebond. This linkage provides a combination of a targeting portion and aneffector portion. Missing from this precedent is the binary targetingaspect of the present invention, since the effector is fully functionalas a single moiety.

[0045] The linkage between the targeting portion and the reconstitutionportion can be a direct chemical crosslink, including disulfide bondsand amide bonds, or it can be mediated by any of the numerousbifunctional linkers known in the literature, including the extensivelist in the Pierce Chemical catalog. And of course, the linkage can beintrinsic to the molecule as synthesized, as in a fusion protein or anRNA aptamer fused to a ribozyme. Although covalent attachment ispreferred, a sufficiently tight non-covalent linkage can also be used,such as a nickel containing moiety and a hexahistidine nickel-chelatingmoiety.

[0046] Many types of chemical entities can participate as targeting orreconstitution portions. In one embodiment, antibodies provide thetargeting portions. For example, two antibodies that bind via theirproximal Fab ends to non-overlapping epitopes on a cell surface receptormay be used as targeting portions of two complementary targetingcomponents. To provide the reconstitution portions, the Fc regions aresuch that they form a discontinuous epitope for which a third antibodyis engineered which recognizes this discontinuous epitope. Thediscontinuous epitope thus is a functional moiety which is able to bindan additional component in turn binds to an effector. Any effector, suchas an enzyme, can then be coupled to the third antibody.

[0047] In addition to standard antibodies, fragments of antibodies orthe related proteins defined above can be used. Other chemical entitiescan also be used to create targeting portions, alone or in combination.As will be recognized by one of ordinary skill in protein bindingassays, an antibody to the EGF receptor can be replaced by EGF itself ora fragment thereof, as an example. Lectins can be targeted tooligosaccharides. Peptide or non-peptide small molecules can berecognized by antibodies or other proteins, as in estrogen receptor andnatural or artificial steroids. Other exemplary targeting portionsinclude oligonucleotides, vitamins, lipids, and drugs that bind tospecific proteins. All such combinations will be familiar to one ofordinary skill in the art of biospecific recognition.

[0048] Sites in Close Proximity

[0049] Any multiplicity of distinct sites located in close proximityform the target. In many applications, the sites in close proximityalready exist and constitute the target for the formation of thefunctional moiety and ultimately effector function. For use in vivo,typically, the sites in close proximity already exist either at cellsurfaces or intracellularly. Thus, in one embodiment, specific epitopeson a cellular structure, either on a cell surface or inside a cell, areused as the distinct sites to anchor the plurality of the targetingcomponents in close proximity.

[0050] Alternatively, an artificial surface can be used to display thedistinct sites. For example, two haptens may be conjugated to a singleprotein or to a latex microsphere in order to capture two targetingcomponents. The distinct sites must be suitable for recognition by thetargeting portions of the targeting components, such as a peptideantigen or hapten recognized by an antibody, or a carbohydraterecognized by a lectin, or a nucleic acid recognized by a complementarybase pairing sequence. Examples of vitamin- oligosaccharide-,carbohydrate-, lipid-, small molecule-binding substances and structuresare known in the art (see e.g. WO 01/02600) and can be used in thepresent method. The plurality of distinct sites can be of the same ordifferent types.

[0051] For example, two protein antigens can be mixed and thenimmobilized on a polystyrene 96-well microplate or on a nitrocellulosemembrane. A protein composed of two different subunits can also providethe distinct sites. Obviously, in all these cases the sites must bespaced sufficiently far apart to avoid steric overlap of the targetingcomponents, but not so far as to eliminate the possibility of assemblinga functional moiety from the attached reconstitution portions. Dependingon the nature of the sites and targeting components, this spacing willvary, but in general it will be in the range of 5 to 50 nm.

[0052] As already noted, there is no intrinsic restriction to only twotargeting components. A trimeric enzyme, such as aspartatetranscarbamoylase, which achieves functionality via contributions fromeach monomer could be adapted to the present purpose. Scaffoldingproteins, including those with pleckstrin homology domains, orcytoskeletal proteins such as actin may also be used to enable more thantwo components to contribute to the assembled functional moiety.

[0053] If an intact cell is targeted, it can exist independently from ahigher structure or it can exist in a multicellular structure orenvironment, e.g., a tissue or an organ of a multicellular organism, amulticellular organism itself or a cell culture. Non-limiting examplesof targetable cells include those from animals, plants, fungi, bacteria,spores, as well as both natural and recombinant cultured cells.

[0054] Prior efforts using antibody-enzyme to activate a prodrug hasfocused on cancer, as a way of increasing the local concentration ofdrug at the tumor site, which is important given the high systemictoxicity of many cancer drugs. The present invention is not limited totreating cancer, however. Almost every drug can benefit from increasedtissue specificity. For example, the invention targeting can be appliedto neuroactive drugs with poor penetration of the blood brain barrier,reducing systemic exposure while achieving high concentration forefficient mass action transfer by targeting activation to sites on thebarrier.

[0055] The cellular structure to be targeted can also be a subcellularstructure, present in an intact cell or as a separated component. Amongthe targetable subcellular structures are nucleus, chromosome,mitochondrion, chloroplast, ribosome, endoplasmic reticulum, Golgiapparatus, lysosome, proteosome, secretory vesicle, microsome, and othervacuoles. Viruses can also be targeted, whether intact or at some stageintermediate stage in the virus life cycle. Intracellular parasites canalso be targeted. The following example illustrates targetingintracellular sites. Two demitopes assemble to form a paratope thatbinds a label. One demitope is fused to an antibody against histones. Alibrary of peptides is conjugated to the complementary second demitope,forming a library of targeting components. If a member of the peptidelibrary induces localization of the targeting component to the nucleus,where the constant anti-histone targeting component is localized, thenthe two demitopes will assemble a functional paratope, enablinggeneration of a signal that identifies the library member as havingnuclear translocation features. A similar strategy can be applied toother subcellular locations, allowing creation of a repertoire ofpeptide reagents, or other chemicals, which induce desiredlocalizations. Defects in cell trafficking may thereby be alleviated.For example, the chloride ion channel encoded by the major cysticfibrosis risk factor gene has nearly normal properties as an ion channelbut does not traffic properly to the apical end of the cell. Agents thatbind to the defective channel and promote its proper localization wouldbe useful therapeutics for cystic fibrosis. Similarly, if the targetingcomponents recognize two transcription factors, then propinquity of thetranscription factors on a chromosome can be used to generate a signalor other effector function.

[0056] In a preferred embodiment, at least one of the plurality ofdistinct sites to be targeted is a marker of a biological pathway, suchas a signal transduction protein. Other important embodiments includesites contributing to a similar biological function such as theribosome, as well as sites that define a stage of cell cycle, a celltype, a tissue type, an organ type, a developmental stage, or a diseaseor disorder.

[0057] In a particularly preferred embodiment, the effector function iscreated on the surface of an intact cell. Effectors that visualize thetargeted cell are thereby localized more specifically. Effectors thatinfluence the cell, e.g. by capturing a radioligand or activating aprodrug, also gain specificity for the targeted cell by this means. Thecell surface sites can be non-overlapping epitopes on a single protein,or the same epitope on different subunits of a single protein. Differentepitopes can also be on proteins that associate by lateral diffusion inthe plane of the membrane. The sites can be native to the cell, orengineered into the cell.

[0058] Illustrative Reconstitution Portions, Functional Moieties andEffector Functions

[0059] A variety of embodiments provide the required reconstitutionportions of the complimentary targeting components. For example, twodemitopes are used to form a paratope as a functional moiety.Preferably, the two demitopes are variable regions of the correspondingheavy and light chains of an antibody. The assembled paratope may thenbind a label; for example, to create an effector function enablingvisualization of the targeted site. Complementary fragments of a nucleicacid aptamer would be useable in the same manner. Even greater varietyof chemical embodiments is useful in instances where the function of theassembled reconstitution portion is simply to display a novel epitopewhere function is recognition by an antibody or analogous protein.Oligosaccharides would be suitable in this aspect, as would certainlipids, and a wide range of haptens (small molecule epitopes). Thediscussion below regarding examples of effector functions will provideadditional information on the use of nonprotein moieties in areconstitution portion.

[0060] The reconstitution portions of complementary targeting componentsassemble into a functional moiety which optionally directly provides aneffector function, such as enzymatic activity, or the functional moietycan bind an intrinsically active effector function moiety such as aradioligand. The functional moiety thus can bind directly or indirectlyan additional moiety that provides the final effector function. Forexample, the assembled functional moiety can bind directly to aneffector, such as an enzyme, or it can bind an epitope, such as biotin,which is attached to the enzyme. Engineered epitopes, such as myc orFLAG can be introduced into the effector enzyme using establishedepitope tagging technology, allowing it to be captured at the site of areconstituted paratope that binds this motif. Further, the assembledfunctional moiety can be coupled to the final effector via a linkingsystem such as a pair. Any suitable linking pair can be used in thissetting, e.g., biotin and avidin; or a FLAG epitope and an antibody thatbinds to the FLAG epitope; or a nickel-containing moiety and ahexahistidine nickel-chelating moiety. For example, the assembledreconstitution portions can bind a ligand conjugated to biotin which inturn is bound to avidin conjugated to an enzyme. Such “sandwich”constructs are well established in the immunological literature.

[0061] Applications

[0062] The method of the invention and the targeting components whichare useful therein can be adapted to a wide variety of applications. Inone illustrative application, the method is applied in gene therapy andrelated research.

[0063] The effector functions are provided by antisense sequences, smallinterfering RNAs, peptide nucleic acids, sequence specific polyamides,or a virus that carries a gene therapy/regulatory payload. The effectorfunction is provided inside a viral particle. A functional receptor sitefor that viral particle is provided on a cell surface by construction ofappropriate proximal sites, or by appropriate design of targetingportions. If the virus is non-infectious to human cells, e.g., an insectvirus, there is a low background for gene vector insertion into humancells. The targeting portions may be antibodies against natural cellsurface receptors, allowing creation of a novel receptor for thexenotropic virus, with the high specificity that arises from binarytargeting. It has been shown that a single chain antibody against hoofand mouth virus, when fused to a cell surface protein (ICAM1),functioned as a novel receptor for the virus, allowing high infectivitywith respect to cells displaying ICAM1 that previously were notsusceptible to the virus (Rieder et al, PNAS (1996) 93:10428-10433).According to the present invention, the paratope recognizing the virusis dissected into two complementary demitopes, each fused to ICAM1 andexpressed by cells. Association of ICAM1 molecules by lateral diffusionin the cell membrane creates a receptor for the virus.

[0064] The invention method may also be used to label specific targets.The effector function is thus a labeling moiety. Exemplary labelingmoieties include chemical, enzymatic, radioactive, phosphorescent,fluorescent, fluorescence-quenching, luminescent and fluorescenceresonance energy transfer (FRET) labels, as well as microspherescontaining dyes of various sorts. The labeling moiety can generate adetectable signal, enhance an existing signal, or quench or weaken anexisting signal. In a preferred embodiment, the labeling moietygenerates an immediately detectable signal, e.g., fluorescencequenching, fluorescence enhancement, or an alteration of NMR spectrum.Examples of such interactions are described in the literature. Forexample, Kranz et al., Proc. Natl. Acad. Sci. USA (1981) 75:5807-5811took advantage of the ability of assembled antibody variable regions toquench the fluorescence of bound fluorescein, by more than 90%, in orderto monitor immunoglobulin recombination and active site formation. Thus,fluorescence quenching is a practical way to monitor reconstitution(active site formation) upon mixing resolved heavy and light chains.Applied to the invention method, the heavy and light chains are thedemitopes and the resulting associated combination is the functionalparatope which, upon binding fluorescein, provides an immediate signal,namely fluorescence quenching. The system of Kranz is adapted to themethod of the present invention by coupling the light chain with a firsttargeting portion and the heavy chain to the second, complementarytargeting portion.

[0065] Rothstein et al., Mol. Immunol. (1983) 20:161-168 used a similarphenomenon, that of fluorescence quenching of p-azophenylarsonate byvarious anti-idiotypic antibodies, as a measure of affinity of theantibody for the dye. The higher the affinity, the more reconstitutedparatopes that are formed, and hence the greater the degree offluorescence. The phenomenon of fluorescence quenching by binding to aparatope is not limited to organic fluorescent molecules. Metal-basedcomplexes may also exhibit quenching upon sequestration in organicenvironments, such as by binding to antibodies or assembled receptors.For example, fluorescence quenching of rubidium complexes occurs uponbinding to antibodies raised against such complexes (Shreder et al., J.Am. Chem. Soc. (1996) 118:3192-3201).

[0066] The immediate signal generated may not necessarily involvequenching of fluorescence, but may, depending on the fluorophore and theparatope, be fluorescence enhancement. For example, as shown by Parkeret al., Biochemistry (1967) 6:3417-3427, antibodies raised againstdansyl-lysine effect a 150-fold enhancement of the fluor's emission uponbinding. Other examples of fluorescence altered by environment include“Quantum Dots” which are clusters of metal atoms. Their fluorescenceproperties are greatly enhanced and tuned to a narrow emission frequencyby appropriate molecular environments (U.S. Pat. No. 6,207,392; Bruchez,et al., Science (1998) 281:2013-2016). Similarly, the NMR properties ofcompounds are often influenced by their environment, specifically theirability to interact with water molecules. For example, a protease assayhas been described which is based on enzymatic cleavage of a gadoliniumcontaining compound to expose the metal to water, thereby drasticallychanging the NMR spectrum (Moats et al., Angew. Chem. Int. Ed. Engl.(1997) 36:726). Similarly, a radio-opaque compound can be captured, forimaging by X-ray or CAT scan technology. A compound showing highreflectivity for ultrasound can likewise be captured.

[0067] In another application, the invention method may be used toinhibit the cell cycle of any intact cell, or otherwise damage or causedeath of said cell or surrounding cells. Any suitable effector can beused for such cell growth inhibitory, damaging or eradication function,such as a radioactive moiety, a toxin or a prodrug which will be coupledto the functional moiety formed by the reconstitution portions. (Fortreating tumors, the targeting portion on each targeting component couldbe an antibody against histone, which is present in cell debris ofnecrotic tumor foci.)

[0068] In another application, the present invention provides for amethod to create an enzymatic activity, for example on a cell surface orin a diagnostic 96-well microplate. In one specific embodiment, thereconstitution portions comprise fragments of an enzyme that areinactive separately but which reconstitute catalytic activity whenassembled as a consequence of co-localization of the targetingcomponents. Examples of such direct formation of enzymatic activity havebeen disclosed (U.S. Pat. Nos. 5,643,734 and 6,270,964). In those priorart examples, the reconstitution event was used to establish whethermoieties attached to the reconstitution portions interact with eachother, rather than to effect targeting.

[0069] Two additional formats for creating enzymatic activity at aspecific target by employing methods similar to those disclosed in U.S.Pat. Nos. 5,643,734 and 6,270,964. In the one format, the tworeconstitution portions are assembled to form an epitope recognitionmoiety, and an enzyme displaying the relevant epitope is thereby boundto the assembled structure, thus providing an enzymatic activity. Avariation of this approach uses a linking pair as described above. Forexample, if the epitope recognized has biotin attached, then the enzymecan be coupled to avidin and thereby associated with the reconstitutedmoiety. Likewise, the assembled reconstitution portions can generate adiscontinuous epitope recognized by an antibody, and the enzyme can becoupled to that antibody. The common feature in all these variations isthat the assembled functional moiety captures an enzyme as an effectorfunction.

[0070] In a second format, one of the reconstitution portions comprisesan exosite modulator for an intact enzyme, which then acts as thecomplementary reconstitution portion. Upon reconstitution, therefore,the effector function is formed directly as the enzymatic activity isimmediately either induced or suppressed. Naturally occurring allostericsites can be used in this way as can non-natural sites, discovered bymolecular biological or combinatorial chemistry techniques (Dennis etal., Nature (2000) 404:465-470). In a variation of this format, one ofthe reconstitution portions comprises a necessary cofactor for anenzyme, such as NADH, and the other demitope comprises an enzyme or afunctional fragment thereof, whereby upon reconstitution, the enzymaticactivity is activated; thus, the reconstitution portions are based onthe approach set forth in the Apo-Enzyme Reactivation Assay (ARIS), inwhich the co-factor FAD is coupled to an analyte (Dosch et al. FreseniusJ. Anal. Chem. (1998) 361:174-178). An enzyme such as glucose oxidasethat uses that cofactor is catalytically inactive if theanalyte-cofactor is sequestered by virtue of binding to an antibody; ifanalyte is introduced which competes for the antibody binding site, thenthe co-factor becomes available and the enzyme is activated.

[0071] In still another variation, one of the reconstitution portionscomprises an inhibitor of an enzyme that serves as the otherreconstitution portion, whereby upon reconstitution, the enzymaticactivity is inhibited. Alternatively, one of the reconstitution portionscan provide a high affinity binding site for a flexible flap thatoccludes or occupies the catalytic site of an enzyme which then servesas the other reconstitution portion, whereby upon reconstitution, theenzymatic activity is activated. Such flaps are well known, e.g., theProtein Kinase C pseudosubstrate sequence that occludes the enzymeactive site until a conformational change in the protein is triggered.Alternatively, one reconstitution portion can supply a pseudosubstatetype inhibitor of activity (Zhong, et al., J. Biol. Chem. (1999)274(48):33913-20). The common feature of all these variations is thatthe activity of an intact enzyme is modulated by the secondreconstitution portion. The created enzymatic activity can be used forany suitable purpose, including generating a signal or activating aprodrug.

[0072] The present invention can also be used to assay an analyte in asample. In one embodiment, at least a first targeting portion recognizesboth the analyte itself and an analog of the analyte immobilized on asurface. Exemplary surfaces include the surface of a microplate, a glassslide, a test tube, a nitrocellulose membrane, a latex or other plasticbead, a colloidal gold particle, a colored particle, a magnetic bead anda quantum dot. The analog can be the analyte itself in an immobilizedform, or a closely related molecule, or it could be any substancecross-reactive with regard to the relevant targeting portion; all ofthese are considered analogs of the analyte for present purposes. Asecond targeting component is able to bind to a site in close proximityto the analyte-occupied site in order to generate a signal. Any signalmoiety can be used, including an enzyme, a radioactive moiety, afluorescent or phosphorescent moiety including microspheres, and an NMRdetectable moiety. Analyte in the sample to be tested competes with theanalyte-occupied site for the first targeting portion. In this manner,any analyte can be assayed which is recognized by at least said firsttargeting portion, by assessing the presence or amount of diminution ofthe signal effected by the sample in comparison to a control lackinganalyte.

[0073] Exemplary analytes include a peptide, a protein, anoligonucleotide, a nucleic acid, a vitamin, an oligosaccharide, acarbohydrate, a lipid, a small molecule and a complex or combinationthereof. The benefit of binary targeting in this instance is maximizedif the analyte is one for which a therapeutic binary targeting system isavailable. That is, in an analytical context, many candidatetherapeutics can be tested against a tumor specimen, for example, todetermine which pair of targeting components is most likely to deliveran effector function with high specificity to the tumor. If a sufficientsample of tumor cells is available, then the candidate pairs can all betested directly, using a signal generating effector. However, if samplequantity is limited, or if it is desired to detect cell free tumorantigen shed into the blood or urine, then the competitive assayanalogous to that described above is more appropriate than a directassay.

[0074] Kits

[0075] The invention also provides kits constructed to provide afunctional moiety and/or an effector function to desired target. Eachkit comprises, in one or more containers, two or more targetingcomponents. Each targeting component comprises a targeting portion and areconstitution portion as described. The reconstitution portions mayprovide an effector function when assembled, or may result in afunctional moiety that is able to couple to an effector function. Thekit may, in the latter case, contain additional components to providethe effector function. Preferably, the kit further comprisesinstructions for use. One preferred kit enables creating an enzymaticactivity at a cell surface by means of binary targeting, for diagnosticimaging or therapeutic treatment of a targeted cell type.

[0076] Illustrative Specific Embodiments

[0077] The specific embodiments described below are intended toillustrate the utility of the present invention, and are not intended tolimit its definition. As will be apparent to one of ordinary skill inthe art of biochemistry, the invention can be embodied in a wide rangeof chemical entitites, all sharing the key feature of multipleindependent targeting components, which provide improved specificity forbringing an effector to a chosen site as compared to the same effectordelivered by a single targeting component.

Preparation A Construction of Targeting Components

[0078] A convenient method to obtain targeting components is described.Single chain antibodies (scFv) are small immunoglobulin-like moleculesthat contain variable regions derived from antibody heavy and lightchains coupled through a linker. These constructs are screened usingstandard phage display techniques (Marks et al., New England J. Med.(1996) 335:730-733). A multiplicity of candidate paratopes is therebyprovided, from which several are selected which bind an effector, suchas biotin or dansyl-lysine. Once appropriate paratopes are identified inthis manner, the linker between the heavy and light chain derivedvariable regions is cleaved at the DNA level to obtain the demitopes,which can be further mutagenized so as to weaken their intrinsicattraction for each other in the absence of linked targeting portions.Similarly, phage display of scFv antibody-like constructs can be used toisolate the targeting portions of the targeting components. A targetingcomponent is then created by chemically linking a targeting portion to ademitope, or by fusing the encoding sequences at the DNA level.

EXAMPLE 1 Imaging of Tumor Cells

[0079] Tumor cells expressing the EGF receptor are imaged followingconstruction of proteins with two subunits, each consisting of twodomains analogous to the constant and variable domains that make up anantibody Fab fragment (represented as C_(H)-V_(H)::C_(L)-V_(L)). TheV_(H)::V_(L) portion that binds antigen is normally called a paratope,and the separated V_(H) and V_(L) portions will be referred to here asdemitopes. Copending application, Ser. No. 10/071,844 filed Feb. 8,2002, incorporated herein by reference, describes replacing the constantdomains (C_(H)::C_(L)) with any proteins (X and Y) that bind to eachother. The paratope (V_(H)::V_(L)) thereby formed by the attachedvariable regions provides a means of detecting the X::Y interaction. Inthe present example, the X and Y portions are comprised of proteins,such as single chain Fv antibodies, that bind non-overlapping epitopeson the EGF receptor. The resulting signal generated as a result of theassembly of a functional paratope provides higher specificity forvisualizing the targeted cell than is feasible with a single targetingcomponent coupled to a signal generating moiety. That is, if X and Yindividually have 1,000:1 preference for their target antigens overother antigens in the body, and are independent in their binding, thenthe X::Y specificity is ˜1,000,000:1. The captured ligand is thefluorescent hapten dansyl-lysine, whose emission is stimulated 150-foldupon binding, allowing detection in an in vitro diagnostic setting.Alternatively, the captured ligand is a compound with a gadolinium atomexposed to water until bound to the assembled paratope. The change inNMR signal when bound as compared to when free in solution therebyenables magnetic resonance imaging of tumor cells inside the body.

[0080] EXAMPLE 2

Drug Delivery

[0081] Similar constructs are used to deliver a therapeutic effector tothe targeted tumor cells. Specifically, the assembled paratope is chosenfor its ability to bind to biotin. A biotin conjugated radioligand or abiotin conjugated prodrug activating enzyme then provides thetherapeutic effector. The prior art has described the pharmacokineticadvantages of introducing small molecule final effectors after thetargeting proteins have maximally localized. Binary targeting preservesthose advantages while enabling higher specificity in the proteintargeting aspect of the technique.

[0082] EXAMPLE 3

Facilitating Virus Entry

[0083] The paratope created by the targeting components constitutes areceptor for an insect virus that is non-infectious to normal humancells. The reconstituted receptor enables a very low background for genetherapy vector insertion into human cells. Feasibility of introducingnovel viral receptors into cells is established by the prior art, inwhich a single chain antibody against hoof and mouth virus was isolated;when fused to a cell surface protein, the antibody functioned as a novelreceptor for the virus, allowing high infectivity for cells thatpreviously were not susceptible to the virus (Rieder et al., Proc. Natl.Acad. Sci. U.S.A. (1996) 93:10428-33).

[0084] EXAMPLE 4

Analysis of Nuclear Localization

[0085] This example illustrates binary targeting of the nucleus usingone targeting component known to bind thereto and a second targetingcomponent containing, as the targeting portion, a peptide whose abilityto localize to the nucleus is to be tested. The method takes advantageof the presence of histones in the nucleus, so that a targetingcomponent where the targeting portion is an antibody to histones isknown to be localized thereto. Thus, an antibody to histones isconjugated to a first demitope. A library of peptides to be tested forlocalization is conjugated to the complementary second demitope astargeting portions. Cells are engineered by standard methods so as toexpress the anti-histone:demitope-1 construct and one member of thepeptide library with its attached demitope-2. The assembled paratope islabeled so as to generate a signal by stimulating fluorescence ofdansyl-lysine. A peptide that induces localization to the nucleusgenerates a signal since the signal is only generated if the peptidebrings the attached demitope-2 into close proximity to demitope-1.

1. A method to provide a functional moiety to a target, which methodcomprises: a) providing two or more targeting components, each of saidtargeting components comprising a targeting portion and a reconstitutionportion, wherein each said targeting portion binds specifically to oneof two or more sites located in close proximity on said target andwherein said reconstitution portions, when brought into close proximity,assemble into a functional moiety at said target; and b) contacting saidtargeting components to an environment comprising said target thereby,inducing assembly of said functional moiety at said target.
 2. Themethod of claim 1, wherein said functional moiety provides an effectorfunction.
 3. The method of claim 2, wherein the effector function is anenzymatic activity.
 4. The method of claim 1, wherein said functionalmoiety directly or indirectly binds an effector function.
 5. The methodof claim 1, wherein the targeting portions are peptides, proteins,oligonucleotides, nucleic acids, vitamins, oligosaccharides,carbohydrates, lipids, small molecules, or a complex or combinationthereof.
 6. The method of claim 1, wherein the reconstitution portionsare peptides, proteins, oligonucleotides, nucleic acids, vitamins,oligosaccharides, carbohydrates, lipids, small molecules, or a complexor combination thereof.
 7. The method of claim 5, wherein the targetingportions are immunoglobulins or fragments thereof.
 8. The method ofclaim 7, wherein the immunoglobulins or fragments are independentlypolyclonal antibodies, monoclonal antibodies, Fab fragments, Fab′fragments, F(ab′)₂ fragments, Fv fragments, diabodies, single-chainantibodies or multi-specific antibodies formed from antibody fragments.9. The method of claim 1, wherein the target is a cellular structure.10. The method of claim 9, wherein the cellular structure is an intactcell.
 11. The method of claim 9, wherein the cellular structure is asubcellular structure.
 12. The method of claim 1, wherein the targetingportions and the reconstitution portions are covalently linked directlyor via a linker.
 13. The method of claim 1, wherein at least one of thetargeting components is a fusion protein.
 14. The method of claim 4,wherein the functional moiety binds to a label.
 15. The method of claim14, wherein the label generates an immediately detectable signal. 16.The method of claim 9, wherein the functional moiety binds to aneffector moiety that is thereby delivered to the cellular structure. 17.The method of claim 10, wherein the functional moiety binds to aneffector moiety that carries out its function on the surface of thecell.
 18. The method of claim 4, wherein the effector moiety that bindsto the functional moiety is a peptide, a protein, an oligonucleotide, anucleic acid, a gene therapy or gene regulatory construct, a virus thatcarries a gene therapy/regulatory payload, a prodrug, a toxin, aradioactive moiety, a NMR detectable ligand or a fluorescent orphosphorescent ligand.
 19. The method of claim 18, wherein the effectormoiety that binds to the functional moiety is an enzyme.
 20. The methodof claim 4, wherein the functional moiety binds an effector directly.21. The method of claim 4, wherein the functional moiety binds aneffector indirectly via a separate linking pair.
 22. The method of claim21, wherein the separate linking pair comprises: a) biotin and avidin orstreptavidin; or b) a FLAG epitope and an antibody that binds to saidFLAG epitope.
 23. The method of claim 1, which is conductedintracellularly.
 24. The method of claim 1, which is conducted in vivo.25. The method of claim 1, wherein the distinct sites located in closeproximity on the target are different regions of a single molecule. 26.The method of claim 1, wherein the distinct sites located in closeproximity on the target are located in separate molecules that canattain close proximity via diffusion, either laterally in the plane of amembrane or in three dimensions.
 27. A kit to provide an effectorfunction to a target, which kit comprises, in one or more containers,two or more targeting components, each of said targeting componentscomprising a targeting portion that binds specifically to one of two ormore distinct sites located in close proximity on a target and areconstitution portion, wherein said reconstitution portions, whenbrought into close proximity, assemble into a functional moiety on saidtarget.
 28. The kit of claim 27, which further comprises an effectormoiety which interacts with or binds to said assembled functionalmoiety.
 29. The kit of claim 27, which further comprises instructionsfor using the targeting components.
 30. A method to create or modify anenzymatic activity at a target which method comprises: a) providing twoor more targeting components, each said targeting component comprising atargeting portion that binds specifically to one of two or more distinctsites located in close proximity at the target and a reconstitutionportion wherein said reconstitution portions, when brought into closeproximity, assemble into a functional moiety, b) contacting saidtargeting components with said target whereby said functional moiety isassembled, wherein said functional moiety functions to create or modifyan enzymatic activity at said target.
 31. The method of claim 30,wherein said functional moiety provides enzymatic activity.
 32. Themethod of claim 31, wherein said functional moiety provides a capturesite for an enzyme or for one or more linking moieties which bind anenzyme.
 33. The method of claim 30, wherein at least one reconstitutionportion comprises a cofactor for at least a second reconstitutionportion which has enzymatic activity in the presence of the cofactor,whereby upon reconstitution, enzymatic activity is created.
 34. Themethod of claim 30, wherein at least one reconstitution portioncomprises an allosteric inhibitor for an enzyme and at least onereconstitution portion comprises an enzyme affected by said allostericinhibitor, whereby upon reconstitution, enzymatic activity is inhibited.35. The method of claim 30, wherein at least one of the reconstitutionportions comprises a high affinity binding site for a flexible flap thatoccludes or occupies the catalytic site of an enzyme and the otherreconstitution portion comprises said enzyme comprising said flap or afunctional fragment thereof, whereby upon reconstitution, the enzymaticactivity is activated.
 36. The method of claim 30, wherein the enzymaticactivity activates a prodrug at the target.
 37. A kit to create anenzymatic activity at a target, which kit comprises, in one or morecontainers, two or more targeting components, each said targetingcomponent comprising a targeting portion that binds specifically to oneof two or more distinct sites located in close proximity on said targetand a reconstitution portion, wherein said reconstitution portions ofsaid targeting components, when brought into close proximity, assembleinto an enzyme or a functional fragment thereof, or into anenzyme-capture site on said target.
 38. The kit of claim 37, whichfurther comprises an enzyme which can be captured by said enzyme capturesite.
 39. The kit of claim 37, which further comprises instructions forusing the targeting components.
 40. A method for assaying an analyte ina sample, which method comprises: a) providing a surface comprising atleast two sites located in close proximity, at least one of said sitescoupled to said analyte or an analog thereof; b) providing at least twotargeting components, comprising targeting portions and complementaryreconstitution portions which when brought into close proximity,assemble into a signal moiety or a binding site for a signal moiety; c)wherein at least one of said targeting components comprises a targetingportion which binds to said analyte, and each other targeting componentcomprises a targeting portion which binds specifically to a site lackingsaid analyte or analog located in close proximity on said surface to thesite coupled to analyte or analog; d) contacting said surface with saidtargeting components in the absence and in the presence of said analyteand detecting the signals, if any, generated by said signal moiety; ande) comparing said signals detected in the absence and in the presence ofsaid analyte to determine the presence and/or amount of said analyte insaid sample.
 41. The method of claim 40, wherein the surface is asurface of a microplate, a glass slide, a nitrocellulose membrane, alatex or plastic bead, a cell, a test tube, a colloidal gold particle, acolored particle, a magnetic bead, or a quantum dot.
 42. The method ofclaim 40, wherein the signal moiety is an enzyme, a radioactive moiety,a visible moiety, an NMR detectable moiety or a fluorescent orphosphorescent moiety.
 43. The method of claim 40, wherein the analyteto be assayed is a peptide, a protein, an oligonucleotide, a nucleicacid, a vitamin, an oligosaccharide, a carbohydrate, a lipid, a smallmolecule or a complex or combination thereof.
 44. The method of claim40, wherein the sample is a biosample.